Antibacterial S-heterosubstituted disulfides

ABSTRACT

Synthetically-derived S,S-heterodisubstituted disulfides that exhibit potent in vitro antibacterial activity against a variety of bacteria, including  Staphylococcus aureus , methicillin-resistant  Staphylococcus aureus  and  Francisella tularensis . The present invention provides compounds, methods and compositions effective to treat microbial/bacterial infections, and, especially, infections arising from bacteria which have developed resistance to conventional antibiotics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior filed InternationalApplication Number PCT/US2008/081125 filed Oct. 24, 2008, which claimspriority to U.S. Provisional Patent Application No. 60/982,403 filedOct. 24, 2007, the contents of which are herein incorporated byreference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.R01AI053281 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF INVENTION

This invention relates to antibiotic compositions. More specifically,this invention relates to antibacterial S-heterosubstituted disulfidecompounds and methods of using these compounds.

BACKGROUND OF THE INVENTION

This invention addresses the severe need for new classes ofantibacterial antibiotics for treatment of bacterial infections, and,especially infections arising from bacteria which have developedresistance to conventional antibiotics, such as methicillin-resistantinfections of Staphylococcus aureus (MRSA), and bacterial speciespresenting significant health concerns due to their morbity andmortality in epidemics, such as Francisella tularensis and Bacillusanthracis.

Antibiotic resistance is a particularly critical health concern and hasincreased dramatically over the past two decades. Antibiotic resistancerefers to the ability of a microorganism to avoid the harmful effects ofan antibiotic by destroying the antibiotic, transporting it out of thecell, or undergoing changes that block its effects. Frequent exposure ofa microorganism to an antibiotic provides conditions favorable to theevolution of antibiotic resistance.

MRSA is a type of Staphylococcus aureus resistant to certain antibioticsincluding methicillin and the more common antibiotics such as oxacillin,penicillin, and amoxicillin. MRSA incidence is on the rise in the U.S.and it has now become recognized as a major community-acquired pathogen.

This invention addresses the severe need for new classes ofantibacterial antibiotics for treatment of methicillin-resistantinfections of Staphylococcus aureus (MRSA) which is of epidemic concerndue to their lethality and difficulty in treating in hospitals and inthe open community.

SUMMARY OF INVENTION

The present invention provides compounds, methods and compositionseffective to treat bacterial infections, and, especially, infectionsarising from bacteria which have developed resistance to conventionalantibiotics. These S-heterosubstituted disulfide compounds,synthetically-derived substances prepared in our laboratory, have novelchemical structures and potent in vitro antibacterial activity againstMRSA. The mechanism of action of these compounds is thought to besimilar, based on the structural and chemical similarities, to that ofpreviously reported N-thiolated beta-lactams (N-alkylthio beta-lactams),which have been shown to create alkyl-CoA disulfides through athiol-disulfide exchange within the cytoplasm ultimately inhibiting typeII fatty acid synthesis. Given this mode of action in inhibiting fattyacid biosynthesis, the antibacterial activity of the compounds, asobserved against Francisella tularensis, Staphylococcus aureus andmethicillin-resistant Staphylococcus aureus, would extend to a widecross-section of Gram-negative and Gram-positive bacteria, including B.anthracis, as well as serving as an antimicrobial against microbes suchas fungi.

In accordance with the present invention there is provided a compoundhaving the formula (I):R₁X₁SSX₂R₂  (I)

Each of X₁ and X₂ of compound (I) can be O, N, NH, or S, with X₁ and X₂not necessarily the same as one-another. For example, X₁ could be O,while X₂ is S. Additionally, each R₁ and R₂ are independently alkyl,alkenyl, alkynyl, aryl, heteroaryl, or heterocyclic. As with X₁ and X₂,R₁ and R₂ need not be the same. The compound can further comprise apharmaceutically acceptable carrier.

In an advantageous embodiment, X₁ and X₂ are NH and R₁ and R₂ can be oneof methyl, ethyl, propyl, isopropyl, butyl, s-butyl, or phenyl. In aparticularly advantageous embodiment R₁ and R₂ are independently propylor phenyl.

In a further advantageous embodiment of the first aspect of theinvention, X₁ and X₂ are S and R₁ and R₂ can be one of methyl, ethyl,propyl, isopropyl, butyl, s-butyl, or phenyl. In a particularlyadvantageous embodiment at least one of R₁ and R₂ are phenyl.

In a still further advantageous embodiments of the first aspect of theinvention, X₁ and X₂ are N and R₁ and R₂ can be one of dimethyl,diethyl, diisopropyl, diallyl, or diisobutyl. In a particularlyadvantageous embodiment at least one of R₁ and R₂ are dimethyl, ordiallyl.

In a second aspect the present invention provides a compound having theformula (I):R₁X₁SSX₂R₂  (I)

In the compound of the second aspect at least one of X₁ and X₂ is O andeach R₁ and R₂ are independently alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclic. If X₁ and X₂ are both O and R₁ and R₂ areboth alkyl, then R₁ and R₂ are not both propyl, isopropyl or butyl. Forexample, if R₁ is propyl, then R₂ can be isopropyl or butyl, but notpropyl.

In an advantageous embodiment, both X₁ and X₂ are O. R₁ and R₂ canindependently be methyl, ethyl, propyl, isopropyl, butyl, s-butyl, orphenyl. In a particularly advantageous embodiment R₁ and R₂ areindependently isopropyl, butyl, or phenyl.

In a third aspect the present invention provides a compound having theformula (I):R₁X₁SSX₂R₂  (I)

In the compound of the third aspect each of X₁ and X₂ can independentlybe O, N, NH, or S and each R₁ and R₂ are independently alkyl, alkenyl,alkynyl, aryl, heteroaryl, or heterocyclic. However, if X₁ and X₂ areboth O and R₁ and R₂ are both alkyl groups, then both of R₁ and R₂ arenot propyl, isopropyl or butyl. In other words, they are not both thesame alkyl group if one of R₁ and R₂ are propyl, isopropyl or butyl. Forexample, if R₁ is propyl, then R₂ can be isopropyl or butyl, but notpropyl. The compound can further comprise a pharmaceutically acceptablecarrier.

In a fourth aspect the present invention provides a compositioncomprising a pharmaceutically acceptable carrier and a compound havingthe formula (I):R₁X₁SSX₂R₂  (I)

In the compound of the fourth aspect each of X₁ and X₂ can independentlybe O, N, NH, or S and each R₁ and R₂ are independently alkyl, alkenyl,alkynyl, aryl, heteroaryl, or heterocyclic.

In a fifth aspect the present invention provides a kit comprising anS-heterosubstituted disulfide and instructions for treating aninfection. In an advantageous embodiment, the S-heterosubstituteddisulfide can be any one of the compounds of the first four aspectsabove.

In a sixth aspect the present invention provides a method of treating orpreventing a microbial infection, comprising administering to a subjectin need thereof an effective amount of a compound comprising anS-heterosubstituted disulfide.

In an advantageous embodiment the method is practiced with anS-heterosubstituted disulfide having the formula (I):R₁X₁SSX₂R₂  (I)

Each of X₁ and X₂ can independently be O, N, NH, or S and each R₁ and R₂are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, orheterocyclic.

In a further advantageous embodiment of the sixth aspect of theinvention the microbe is a bacterium. The bacterium can be a bacteriumfrom a genus including Staphylococcus, Bacillus, Francisella,Micrococcus, Streptococcus, Neisseria, Streptomyces, and Mycobacterium.In a particularly advantageous embodiment the bacteria ismethicillin-susceptible Staphylococcus aureus, methicillin-resistantStaphylococcus aureus, Francisella tularensis or Bacillus anthracis.Administering can be carried out by a route selected from the groupconsisting of oral, intravenous, and topical.

In a seventh aspect the present invention provides a method of treatingor preventing a bacterial infection, by administering to a subject inneed thereof an effective amount of a compound having the formula (I):R₁X₁SSX₂R₂  (I)

Each of X₁ and X₂ can independently be O, N, NH, or S and each R₁ and R₂are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, orheterocyclic.

In an advantageous embodiment of the seventh the bacterium is from thegenus Staphylococcus, Bacillus, Francisella, Micrococcus, Streptococcus,Neisseria, Streptomyces, or Mycobacterium. In a particularlyadvantageous embodiment the bacteria is methicillin-susceptibleStaphylococcus aureus, methicillin-resistant Staphylococcus aureus,Francisella tularensis or Bacillus anthracis. Administering can becarried out by a route selected from the group consisting of oral,intravenous, and topical.

In a seventh aspect the present invention provides a method ofinhibiting the growth of Francisella tularensis, Staphylococcus aureusor methicillin-resistant Staphylococcus aureus by the step of contactingthe Francisella tularensis, Staphylococcus aureus ormethicillin-resistant Staphylococcus aureus with an effective amount ofa compound comprising an S-heterosubstituted disulfide.

In a eighth aspect the present invention provides a method of inhibitingthe growth of Francisella tularensis, Staphylococcus aureus ormethicillin-resistant Staphylococcus aureus by the step of applying theS-heterosubstituted disulfide to a surface that may come into contactwith the Francisella tularensis, Staphylococcus aureus ormethicillin-resistant Staphylococcus.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a bar graph illustrating the bioactivity of compounds I-XV andpenicillin against Staphylococcus aureus and methicillin-resistantStaphylococcus aureus. Bioactivity was tested by agar dilution assay.

FIG. 2 is an illustration of the general formula for anS,S-Heterodisubstituted Disulfide (“S-Heterosubstituted Disulfide”).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides compounds, methods and compositionseffective to treat bacterial infections, and, especially, infectionsarising from bacteria which have developed resistance to conventionalantibiotics. These S-heterosubstituted disulfide compounds,synthetically-derived substances prepared in our laboratory, have novelchemical structures and potent in vitro antibacterial activity againstMRSA. New antibacterial compounds are in urgent need, particularly thoseeffective against MRSA. The S-heterosubstituted disulfides of theinvention may represent a critical advance in the battle againstinfectious disease. It is therefore important to continue thedevelopment of these compounds, and to fully evaluate and exploit theirpotential as a new generation of anti-microbials. The compounds of thepresent invention (also referred to herein as S,S-HeterodisubstitutedDisulfides and S-Heterosubstituted Disulfides) exhibit antibacterialactivity against bacteria such as Staphylococcus aureus,methicillin-resistant Staphylococcus aureus and Francisella tularensis.

The present invention provides compounds having the general formula:R₁X₁SSX₂R₂  (1)

-   -   or alternatively represented as:

where X can be O, N, NH, or S (in any combination), and R can includesthe groups of alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic(such as a beta-lactam or 2-oxazolidinone), and so on, as is presentedmore fully below. The compounds may or may not be symmetrical in theirsubstitution, so by way of example, CH₃O—S—S—OCH₃ could be one versionof a symmetrical compound, while additional compounds can be of the formCH₃O—S—S—OCH₂CH₃ or CH₃O—S—S—NHCH₃, thus adopting asymmetric heterosubstituted groups.

The evidence indicates that the compounds block bacterial growth andreplication by inhibiting fatty acid biosynthesis in the microorganism,though the details of the mode of action remains to be fully elucidated.The compounds of the present invention may therefore be used to prevent,alleviate, or cure diseases caused by pathogens whose growth isinhibited by these compounds. The instant compounds are particularlyactive against bacteria and bacteria-like microorganisms. They aretherefore proposed for use in human and veterinary medicine, for theprophylaxis and chemotherapy of local and systemic infections caused bythese pathogens. As used herein, the terms “treat”, “treating”, and“treatment” include therapy that alleviates one or more symptoms causedby the infection, or that cures the underlying infection. As usedherein, the terms “prevent”, “preventing”, and “prevention” includeprophylaxis, complete prevention, or delaying onset of the infection ordelaying onset of one or more symptoms caused by the infection.Optionally, the method further comprises identifying the human ornon-human subject as one suffering from a specific or non-specificbacterial infection. The presence of bacterial infection can bedetermined using methods known to those of ordinary skill in the art ofclinical diagnosis of disease (e.g., infectious disease).

The invention further encompasses methods for inhibiting the growth ofbacteria by contacting the bacteria with an effective amount of thecompounds of the invention in vitro or in vivo, or by applying thecompound to a substrate (surface) likely to come in contact with thebacteria, such as a work surface, table, surgical instrument, implant orother device to be placed in or on the body (i.e., foreign object to beinserted into a subject, such as a stent, catheter, access port,intravenous delivery tube, heart valve, dental implant,electro-mechanical device, prosthetic device, glucose sensor, orstabilizing device such as orthopedic nails and pins), eating or cookingutensil, etc. Because of their powerful antibacterial properties, thepresent compounds may also be used to supplement feed for animals.

In addition, the compounds of the present invention that exhibitantibacterial activity may also be used as medicaments, and also assubstances for preserving inorganic and organic materials, especiallyorganic materials of all kinds, for example, polymers, lubricants,paints, fibers, leather, paper, timber, foodstuffs, and water. Forexample, these compounds can be covalently bonded to the polymer.

DEFINITIONS

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents,“administration” and its variants are each understood to includeconcurrent and sequential introduction of the compound or prodrugthereof and other agents.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician. In reference to a microbial infection, an effectiveamount comprises an amount sufficient to kill or inhibit the growth andreplication of the microbe. In some embodiments, an effective amount isan amount sufficient to delay development. In some embodiments, aneffective amount is an amount sufficient to prevent or delay occurrenceand/or recurrence. An effective amount can be administered in one ormore doses.

As used herein, “treatment” refers to obtaining beneficial or desiredclinical results. Beneficial or desired clinical results include, butare not limited to, any one or more of: alleviation of one or moresymptoms, diminishment of extent of infection, stabilization (i.e., notworsening) of the state of infection, preventing or delaying spread ofthe disease (such as pathogen growth or replication), preventing ordelaying occurrence or recurrence of the disease, delay or slowing ofdisease progression and amelioration of the disease state. The methodsof the invention contemplate any one or more of these aspects oftreatment.

A “subject in need of treatment” is a mammal with a microbial infectionthat is life-threatening or that impairs health or shortens the lifespanof the mammal.

A “pharmaceutically acceptable” component is one that is suitable foruse with humans and/or animals without undue adverse side effects (suchas toxicity, irritation, and allergic response) commensurate with areasonable benefit/risk ratio.

A “safe and effective amount” refers to the quantity of a component thatis sufficient to yield a desired therapeutic response without undueadverse side effects (such as toxicity, irritation, or allergicresponse) commensurate with a reasonable benefit/risk ratio when used inthe manner of this invention.

A “pharmaceutically acceptable carrier” is a carrier, such as a solvent,suspending agent or vehicle, for delivering the compound or compounds inquestion to the animal or human. The carrier may be liquid or solid andis selected with the planned manner of administration in mind. Liposomesare also a pharmaceutical carrier. As used herein, “carrier” includesany and all solvents, dispersion media, vehicles, coatings, diluents,antibacterial and antifungal agents, isotonic and absorption delayingagents, buffers, carrier solutions, suspensions, colloids, and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

The compounds of the present invention include all hydrates and salts ofthe S-heterosubstituted disulfides that can be prepared by those ofskill in the art. Under conditions where the compounds of the presentinvention are sufficiently basic or acidic to form stable nontoxic acidor base salts, administration of the compounds as salts may beappropriate. Examples of pharmaceutically acceptable salts are organicacid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganicsalts may also be formed, including hydrochloride, sulfate, nitrate,bicarbonate, and carbonate salts. When reference is made to a compoundor administering a compound, the recitation of the compound includes apharmaceutically acceptable salt thereof.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a patient, such as ahuman patient, in a variety of forms adapted to the chosen route ofadministration, e.g., orally or parenterally, by intravenous,intramuscular, topical, or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. The amount of active compound in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water or other suitablesolvent, optionally mixed with a nontoxic surfactant. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, triacetin,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredientpresenting the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied inpure-form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from adsorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of the invention to the skinare disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art (U.S. Pat. No.4,938,949 (Borch et al.)).

Accordingly, the invention includes a pharmaceutical compositioncomprising a compound of the present invention as described above, or apharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, comprising an amount ofone or more compounds effective to treat a bacterial infection, are apreferred embodiment of the invention.

The following definitions are used, unless otherwise described. Halo isfluoro, chloro, bromo, or iodo. “Alkyl,” “alkoxy,” etc. denote bothstraight and branched groups, but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical, a branchedchain isomer such as “isopropyl” being specifically referred to. “Aryl”denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radicalhaving about nine to ten ring atoms in which at least one ring isaromatic. “Heteroaryl” encompasses a radical attached via a ring carbonof a monocyclic aromatic ring containing five or six ring atomsconsisting of carbon and one to four heteroatoms each selected from thegroup consisting of non-peroxide oxygen, sulfur, and N(R_(R)) whereinR_(x) is absent or is hydrogen, oxo, alkyl, phenyl or benzyl, as well asa radical of an ortho-fused bicyclic heterocycle of about eight to tenring atoms derived therefrom, particularly a benz-derivative or onederived by fusing a propylene, trimethylene, or tetramethylene diradicalthereto. “Heteroalkyl” encompasses the replacement of a carbon atomwithin an alkyl chain with a heteroatom; e.g., replacement with anelement other than carbon such as N, S, or O, including both an alkylinterrupted by a heteroatom as well as an alkyl substituted by aheteroatom.

It will be appreciated by those skilled in the art that compounds of theinvention having one or more chiral center(s) may exist in and beisolated in optically active and racemic forms. Some compounds mayexhibit polymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis, from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase), and how to determine antibacterial activity using thetests described herein, or using other tests which are well known in theart. Specific and preferred values listed below for radicals,substituents, and ranges, are for illustration only; they do not excludeother defined values or other values within defined ranges for theradicals and substituents.

Specifically, unless otherwise indicated, “alkyl” can include, forexample, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,pentyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl, tetradecyl or pentadecyl; “alkenyl” can include vinyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl,5-heptenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl,6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl,4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl;1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl,6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl,1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl,6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl,11-dodecenyl, 1-tridecenyl, 2-tridecenyl, 3-tridecenyl, 4-tridecenyl,5-tridecenyl, 6-tridecenyl, 7-tridecenyl, 8-tridecenyl, 9-tridecenyl,10-tridecenyl, 11-tridecenyl, 12-tridecenyl, 1-tetradecenyl,2-tetradecenyl, 3-tetradecenyl, 4-tetradecenyl, 5-tetradecenyl,6-tetradecenyl, 7-tetradecenyl, 8-tetradecenyl, 9-tetradecenyl,10-tetradecenyl, 11-tetradecenyl, 12-tetradecenyl, 13-tetradecenyl,1-pentadecenyl, 2-pentadecenyl, 3-pentadecenyl, 4-pentadecenyl,5-pentadecenyl, 6-pentadecenyl, 7-pentadecenyl, 8-pentadecenyl,9-pentadecenyl, 10-pentadecenyl, 11-pentadecenyl, 12-pentadecenyl,13-pentadecenyl, 14-pentadecenyl; “alkoxy” can include methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,hexoxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy,tridecyloxy, tetradecyloxy, or pentadecyloxy; “alkanoyl” can includeacetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl,nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,or pentadecanoyl; “cycloalkyl” can include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. “Aryl” can includephenyl, indenyl, 5,6,7,8-tetrahydronaphthyl, or naphthyl. “Heteroaryl”can include furyl, imidazolyl, tetrazolyl, pyridyl, (or its N-oxide),thienyl, pyrimidinyl (or its N-oxide), indolyl, or quinolyl (or itsN-oxide). “Heterocyclic” can include beta-lactam or 2-oxazolidinone.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Preferably,the compound of the invention (S-heterosubstituted disulfides) isadministered in an isolated or pure form.

As used in this specification, the singular forms “a”, “an”, and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a microorganism” includes more thanone such microorganism. A reference to “a cell” includes more than onesuch cell, and so forth. A reference to “a compound” includes more thanone such compound.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology that arewithin the skill of the art. Such techniques are explained fully in theliterature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to MolecularCloning (1984); the series, Methods In Enzymology (S. Colowick and N.Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hameset al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H.Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification Principles and Practice (2nded., Springer-Verlag); and PCR: A Practical Approach (McPherson et al.Eds. (1991) IRL Press)), each of which are incorporated herein byreference in their entirety.

This invention addresses the severe need for new classes ofantibacterial antibiotics for treatment of bacterial infections, and,especially infections arising from bacteria which have developedresistance to conventional antibiotics, such as methicillin-resistantinfections of Staphylococcus aureus (MRSA), and bacterial speciespresenting significant health concerns due to their lethality inepidemics, such as Francisella tularensis and Bacillus anthracis. Theinvention is now further described by way of the following examples,which, while illustrative of the invention, are not intended aslimitations to the scope. It should be understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application.

Example 1 Synthesis and Antibacterial Activity of S-HeterosubstitutedDisulfides

The target heterosubstituted disulfides are prepared by thiolationaccording to the following reaction:

Representative synthesized compounds are presented in Table 1.

Bioactivity was tested by agar dilution assay against Staphylococcusaureus and methicillin resistant Staphylococcus aureus (Table 2). Thecompounds of the invention were evaluated against Staphylococcus aureusand methicillin-resistant Staphylococcus aureus bacteria strains (i.e.strains ATCC-849 and ATCC-919, respectively) by determining the minimuminhibitory concentration (MIC, μg/ml) of each compound with respect toeach strain. The MIC, or minimum inhibitory concentration, is the lowestconcentration of antibiotic which inhibits growth of the test organism,and was determined for the synthesized compounds by the agar dilutionmethod. A control plate with a reference drug, penicillin G, was used asthe control.

As can be seen in the table, many of the synthesized compounds were aseffective, or more effective, against MRSA than the Penicillin Gcontrol, even if they were not as effective as the control againstStaphylococcus aureus.

While not intending to be bound to a particular theory, the mechanism ofaction of these compounds is thought to be similar, based on thestructural and chemical similarities, to that of previously reportedN-thiolated beta-lactams (N-alkylthio beta-lactams), which have beenshown to create alkyl-CoA disulfides through a thiol-disulfide exchangewithin the cytoplasm ultimately inhibiting type II fatty acid synthesis.These compounds may also be characterized as essentially “stable”analogues of natural disulfides obtained from garlic and onions, whichare fatty acid biosynthesis inhibitors. Given this mode of action ininhibiting fatty acid biosynthesis, the antibacterial activity of thecompounds, as observed against Francisella tularensis, Staphylococcusaureus and methicillin-resistant Staphylococcus aureus, would extend toa wide cross-section of Gram-negative and Gram-positive bacteria,including B. anthracis, as well as fungi.

These structurally-simple disulfides serve as effective antibacterialsfor drug-resistant staph infections.

Bioactivity was tested by agar dilution assay against the live vaccinestrain of Francisella tularensis using a broth dilution technique. Theresults of the test are presented in Table 3. As can be seen from thetable, most of the twenty synthesized compounds were effective againstthe strain at dilutions lower than the ciprofloxacin control.

TABLE 1 Antibacterial S-Hetero Substituted Disulfides

TABLE 2 Anti-Staphylococcus aureus Activity Data MIC (μg/mL) SubstrateCompound R S.A.¹ MRSA² Alcohol I propyl 32 32 II isopropyl 1 0.5 IIIbutyl 0.25 8 IV s-butyl 16 8 V phenyl 2 2 1° Amine VI propyl 8 4 VIIisopropyl 32 32 VIII butyl 8 32 IX s-butyl 16 16 X phenyl 4 4 Thiol XIpropyl 32 32 XII isopropyl 32 32 XIII butyl 64 32 XIV s-butyl 32 64 XVphenyl 8 8 2° Amine XVI dimethyl 0.25 0.5 XVII diethyl 2 2 XVIIIdiisopropyl 2 1 XIX diallyl 1 0.5 XX diisobutyl 16 16 Control PenicillinG 0.125 16 ¹S.A: Staphylococcus aureus (ATCC-849) ²MRSA: MethicillinResistant Staphylococcus aureus (ATCC-919)

TABLE 3 Anti-tularaemia Activity Data Substrate Compound R MIC (μg/mL)*Alcohol I propyl 16 II isopropyl 4 III butyl 4 IV s-butyl 16 V phenyl 11° Amine VI propyl 16 VII isopropyl 2 VIII butyl 4 IX s-butyl 8 X phenyl0.5 Thiol XI propyl 1 XII isopropyl 8 XIII butyl 4 XIV s-butyl 16 XVphenyl 2 2° Amine XVI dimethyl 8 XVII diethyl 16 XVIII diisopropyl 32XIX diallyl 16 XX diisobutyl 1 Control Ciprofloxacin 16 *MIC valuesagainst Live Vaccine Strain of Francisella tularensis using brothdilution technique.

TABLE 4 Antibacterial S-Hetero Substituted Disulfides

All references cited in the present application are incorporated intheir entirety herein by reference to the extent not inconsistentherewith.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween. Now that theinvention has been described,

What is claimed is:
 1. A compound having the formula (I):X₁SSX₂  (I) wherein each X₁ and X₂ are concurrently O—R₁, orN-(1-phenylethyl)aniline; and wherein R₁ is s-butyl, phenyl,


2. The compound according to claim 1 further comprising apharmaceutically acceptable carrier.
 3. A composition comprising acompound having the formula (I):X₁SSX₂  (I) wherein each X₁ and X₂ are concurrently O—R₁, orN-(1-phenylethyl)aniline; wherein R₁ is phenyl,

and a pharmaceutically acceptable carrier.
 4. A method for treating amicrobial infection, comprising administering to a subject in needthereof an effective amount of a compound comprising anS-heterosubstituted disulfide or hydrate or salt thereof, wherein theS-heterosubstituted disulfide has the formula (I):X₁SSX₂  (I) wherein each X₁ and X₂ are concurrently O—R₁, orN-(1-phenylethyl)aniline; and wherein R₁ is s-butyl, phenyl,

wherein the microbe is a bacterium from a genus selected from the groupconsisting of Staphylococcus, Bacillus, Francisella, Micrococcus,Streptococcus, Neisseria, Streptomyces, and Mycobacterium.
 5. The methodof according to claim 4, wherein the bacteria is methicillin-susceptibleStaphylococcus aureus or methicillin-resistant Staphylococcus aureus. 6.The method according to claim 4, wherein the bacteria is Francisellatularensis.
 7. The method according to claim 4, wherein the bacteria isBacillus anthracis.
 8. The method according to claim 4, wherein theadministering is carried out by a route selected from the groupconsisting of oral, intravenous, and topical.
 9. A method for inhibitingthe growth of Francisella tularensis, Staphylococcus aureus ormethicillin-resistant Staphylococcus aureus comprising the step ofcontacting the Francisella tularensis, Staphylococcus aureus ormethicillin-resistant Staphylococcus aureus with an effective amount ofa compound comprising an S-heterosubstituted disulfide or hydrate orsalt thereof, wherein the S-heterosubstituted disulfide has the formula(I):X₁SSX₂  (I) wherein each X₁ and X₂ are concurrently O—R₁, orN-(1-phenylethyl)aniline; and wherein R₁ is s-butyl, phenyl,